The lactic acid bacterium Lactococcus lactis is used in milk fermentations world wide in the dairy industry to produce a variety of cultured dairy products. Phage infections can ruin the fermentation by inactivating the inoculated cultures. Phages are the major cause of fermentation failures during the manufacture of these cultured dairy products. There is thus a permanent need in the art for L. lactis starter cultures to perform at a high level of consistency and efficiency.
Phages
Lactococcal phages are characterized by having relatively short latent periods and relatively large burst sizes. They are the major cause of fermentation failure leading to production loss in the dairy industry. Lactococcal phages are currently divided into eight distinct groups of which three groups namely “936”, “c2” and “P335” are responsible for the vast majority of phage attacks in industrial fermentations. The genomes of the phages within one single group are highly conserved except for the P335 group.
Industrial fermentations are carried out in large fermentation vats in a non-sterile environment. Prior to fermentation, the ingredients are usually pasteurized. However, the phages are often resistant to the pasteurization process. Presence of phages can lead to variations in flavor and texture of the fermented dairy product or even loss of the entire production with serious economical loss as a consequence. The dairy industry is therefore using a variety of methods in limiting phage attacks. Such approaches include e.g. improved disinfection processes, rotation of starter cultures and application of phage resistant starter strains.
Phage Defense Mechanisms
During evolution L. lactis has developed a series of defense mechanisms against phage attacks. These naturally occurring phage resistance mechanisms (φrm) has been studied extensively and also applied in industrial starter cultures. Most of the naturally occurring φrms are found on plasmids and they are classified into four groups according to their mode of action: 1) adsorption inhibition, 2) blocking of phage DNA injection, 3) restriction/modification systems (R/M) and 4) abortive infection mechanisms (Abi). Among these defense mechanisms, the Abi systems are considered to be the most powerful due to their diverse mode of action and efficiency against the most common phages.
Abi Mechanisms
Abi mechanisms function in the phage life cycle subsequent to the injection of phage DNA into the bacterial cell—typically after expression of early phage genes. As a consequence, the phage lytic cycle is terminated and usually the host dies. Very few viable phage progeny are thus released and the phenotypic outcome is a reduction in the number and size of plaques and thus a reduction of the severity of the phage infection.
To date, twenty-two lactococcal Abi systems have been isolated. These Abi systems target one, two or all three groups of the common phage species 936, c2, P335 with varying efficiency (EOP values from 10−1 to <10−8) (FIG. 1).
Most of the isolated Abi systems are found on plasmids of which many are conjugative. By sharing the φrms within the bacterial population, conjugation thus provides an adaptation strategy to the phage containing dairy environment. Only a few abi mechanisms have been isolated from the chromosome of L. lactis. This may partly be due to the fact that it is generally easier to isolate genes present on plasmids compared to isolation of genes present on chromosomes. The procedure used in the present invention to isolate a φrm from the chromosome of L. lactis can be used to identify other φrms on the bacterial chromosome.
By isolating spontaneous phage resistant mutants with a similar phenotype with regards to efficiency against a range of phage species it is probably possible to identify strains expressing the abi without having to use genetic modification. Using this method, non-GMO phage resistant strains can thus be isolated. Use of non-GMO starter cultures may be an advantage in some case, in particular in relation to the fact that the legislation in some countries does not allow use of GMO. Furthermore, some consumers tend to prefer non-GMO derived products.
The point of interference with the phage life cycle has been determined to some degree for most of the Abi mechanisms:                AbiA, AbiF, AbiK, AbiP, AbiR, and AbiT apparently interfere with phage DNA replication.        AbiC apparently interfere with capsid production.        AbiE, AbiI, and AbiQ apparently interfere with phage packaging.        AbiB is apparently an RNase.        AbiD1 seems to interfere with a phage RuvC-like endonuclease.        AbiU apparently delays phage transcription.        AbiZ apparently causes premature lysis of the infected cell.        
These very diverse modes of action are most likely the reason for the very low degree of protein homology that exists between the different Abi mechanisms.
Though the point of action in the phage life cycle has been determined, the phage protein interacting with the Abi mechanism has only been identified in AbiA, AbiD1, AbiK and AbiP. An increasing number of phage genomes are being sequenced providing a bulk of sequence data in which numerous putative proteins are found. However, experimental evidence for the function of these proteins are lacking behind.
Several phage resistant strains of L. lactis have been constructed by introducing abi systems in phage sensitive industrial starter cultures. However, extensive use of these bacterial cultures leads to problems with emergence of phage mutants capable of overcoming the introduced abi systems.
The evolutionary “arms race” between phage mutants and bacterial φrms means that there is a constant need in the art for identifying novel natural φrms. There is a particular need in the art for finding novel Abi-mechanisms that interact with previously unknown targets in the phage. Furthermore there is a need in the art for novel Abi-mechanisms in Lactococcus bacteria that do not classify as GMO. Finally there is a need in the art for identifying φrms that provide efficient protection against phages.